Single-Channel Currents of the Permeability Transition Pore from the Inner Mitochondrial Membrane of Rat Liver and of a Human Hepatoma Cell Line

Abstract

Single-channel currents were recorded from inner mitochondrial membranes of HepG2 hepatoma cells and of normal rat liver cells by means of patch-clamp techniques. The current events showed variable amplitudes of up to 1.1 ± 0.2 nS (n = 35) at room temperature (24 °C) and of up to 1.5 ± 0.2 nS (n = 10) at 34 °C including large numbers of subconductance states. Voltages of -40 mV and below closed the channels usually with a delay of about 2 min. Increasing Ca²⁺ concentrations activated the channels, whereas cyclosporin A (100 nM) blocked. The concentration-response relation for the Ca²⁺-effect could be fitted best using an EC₅₀ of 10 µM and a Hill coefficient of 1.5. Taken together the results indicate that we recorded from the permeability transition pore (PTP). As PTP activity may be related to apoptosis we tested if lysate from differently treated T-lymphocytes (Jurkat cells) was able to induce PTP activity in HepG2 cells. Lysate of untreated cells completely abolished the activity at a Ca²⁺ concentration of 18 nM (buffered by EGTA), i.e. three orders of magnitude below the EC₅₀. Under these conditions the lysate is not likely to contain stable factors that could open the PTP. Preliminary experiments show PTP activity in CD95-activated lysate.

Hypoxia Increases Activity of the BK-Channel in the Inner Mitochondrial Membrane and Reduces Activity of the Permeability Transition Pore

“…We recorded single-channel current of the PTP from rat liver mitochondria in isotonic solutions with 1 mM DTN (n=4) or bubbled for 2 h by N 2 (n=4) and the respective controls before and after hypoxia. The PTP showed, as usual, very large conductance (>1 nS) with several sub-conductance states and could be inhibited by the specific inhibitor cyclosporine A (CSA; see [18] for the complete concentration-response curve) but was neither affected by 100 nM Ibtx (n=4, not shown), nor spontaneously on its own (tested for more than 24 min). Conductance and appearance of sub-states was unchanged in hypoxic solutions as compared with control solution (both containing 200 µM Ca 2+ for activating the PTP, Fig.…”

Inhibitory modulation of the mitochondrial permeability transition by minocycline

“…It must be noted, however, that a mitoplast constitutes an artificial system in that the outer membrane is missing and, secondly, the mitochondrial membrane potential is lost. Nevertheless, mitoplasts have been proved to be useful tools to study successfully biophysical questions and direct pharmacological interactions with the channel proteins in the inner membrane [33,34]. Additionally, it is remarkable that inhibition of the mPTP can be observed at relatively high Ca were reported before [33].…”

“…Inward currents always deflect downward. The probability that the channel is in an open state (P o ) was determined by all-points analysis according to Loupatatzis et al [34]. To the equation for fitting the doseresponse curve by Michaelis-Menten kinetics, a term A was added accounting for a seemingly incomplete blockade of the mPTP in the early phase of the experiment.…”

“…The predominant channel of such patches showed a single-channel conductance of more than 1 nS, could be dose-dependently blocked by CsA, and was characterized by multiple substrates [34]. Patches demonstrating corresponding single-channel behavior were transferred to a pipe of the flow system that contained test solution with different concentrations of MC.…”

Activation of the Permeability Transition Pore by Bax via Inhibition of the Mitochondrial BK Channel

“…Patch-clamp techniques have demonstrated that channel events with characteristic features, such as sensitivity to Ca 2+ and cyclosporin A (CsA), and extraordinarily high single-channel conductance (γ) of more than 1 nS, combined with several substates of lower conductance, could be detected in mitoplasts (i.e., tiny vesicles of the inner membrane [IM] devoid of OM) [12][13][14]. Unfortunately, however, the molecular nature of the mtPTP has remained enigmatic.…”